System, Method and Apparatus for Purifying Biological Fluids Such as Blood and Constituents Thereof

ABSTRACT

Provided herein is an innovative hemoperfusion system and method including a thermal-fused broad-spectrum biocidal iodinated interactive polymer for the treatment of biological contaminants in body fluids. In one exemplary embodiment, the system and method utilizes a Triosyn® thermal fused broad-spectrum iodinated interactive polymer, included in a hemoperfusion column, for devitalizing high levels of cell-free microorganisms in whole blood and biological fluids in relation with the characterization of blood cells viability and function post-treatment

CLAIM OF PRIORITY/CROSS REFERENCE OF RELATED APPLICATIONS)

This application is a continuation of U.S. application Ser. No.12/324,128, filed Nov. 26, 2008 which is a divisional of U.S.application Ser. No. 10/938,693, filed on Sep. 10, 2004, which claimsthe benefit of priority of U.S. Provisional Application No. 60/501,780,filed Sep. 10, 2003. The earlier disclosures of the aforesaid patentapplication are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The funding for work described herein was provided by the FederalGovernment, under a grant from the U.S. Army Medical Research andMaterial Command. The Government may have certain rights in theinvention.

FIELD OF THE INVENTION

This invention relates to biological fluid purification and particularlyto the purification of blood and constituents thereof.

BACKGROUND OF THE INVENTION

The development of an effective purifying process active against a broadspectrum of infectious agents could improve the safety level of bloodproducts by eliminating infectious agents that may remain undetected bycurrent procedures or new infectious agents for which no screening testhas been developed yet.

In the context of emergency conditions such as military operations onthe battlefield and peacekeeping operations where supplying, conservingand testing of safe blood becomes a real challenge, the development of atechnology that would provide real-time blood purification whenwithdrawing blood from a donor would represent an important progress.

In the context of infectious diseases, the development of a technologythat would provide real-time blood purification would enable blood to bewithdrawn form a patient, purified and provided back to the patientattenuating or curing the disease.

It is known that iodinated resins such as disclosed and claimed in U.S.Pat. No. 5,639,452, which issued to Pierre Jean Messier on Jun. 17, 1997and is entitled “Iodine/resin disinfectant and a procedure for thepreparation thereof” can be used to purify biological fluids such asblood. It has been found however that depending on the degree ofinfection and or usage of the given purification system, situationsexist in which the use of such resins damage some or all of the bloodconstituents before effective purification is accomplished, the contacttime between the resin and the cells/substrate being one of thedetermining factors in terms of preservation of cellular integrity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the Triosyn® Integrated Blood Filtration System.

FIG. 2 depicts the correlation between residual iodide and methemoglobinlevels after filtration.

FIG. 3 depicts the progression of MS2 reduction versus residual iodidelevels in blood over a 6-month testing period.

FIG. 4 depicts the progression of E. coli reduction versus residualiodide levels in blood over a 6-month testing period.

FIG. 5 depicts the progression of S. aureus reduction versus residualiodide levels in blood over a 6-month testing period.

FIG. 6 depicts the progression of MS2 reduction versus methemoglobinlevels in blood over a 6-month testing period.

FIG. 7 depicts the progression of E. coli reduction verus methemoglobinlevels in blood over a 6-month testing period.

FIG. 8 depicts the progression of S. aureus reduction versusmethemoglobin levels in blood over a 6-month testing period.

FIG. 9 depicts the Triosyn “J” filter prototype used for plateletdecontamination. Characteristics of the “J” filter: Number of capsulesections: 1. Length of each capsule sections: 5′. Total length ofcapsule: 5′. Capsule inner diameter: 0.625′. Number of diffusers: 0.Triosyn type: iodine component can be 30-80% concentration range,preferably 35-50%. Volume of Triosyn: 25 g. Triosyn size: 500 microns.

FIG. 10 depicts the evolution of bacterial load over time in treated vs.non-treated platelets.

FIG. 11 depicts the evolution of Staphylococcus aureus concentrationover time in treated vs. non-treated platelets.

FIG. 12 depicts the average evolution of Staphylococcus aureusconcentrations over time in treated vs. non-treated platelets, wheren=6.

FIG. 13 depicts the correlation between Lactate Dehydrogenase (LDH)levels and platelet counts in control samples over a six-day period.

FIG. 14 depicts the correlation between Lactate Dehydrogenase (LDH)levels and platelet counts in treated samples over a six-day period.

FIG. 15 depicts the correlation between Lactate and pH levels in controlsamples over a six-day period.

FIG. 16 depicts the correlation between Lactate and pH levels in treatedsamples over a six-day period.

FIG. 17 depicts the characteristics of the “BB” filter. Characteristicsof the “BB” filter: Number of capsule sections: 5. Length of eachcapsule sections: 2.75′. Total length of capsule: 13.75′. Capsule innerdiameter: 0.75′. Number of diffusers: 4. Triosyn type: iodine componentcan be 30-80% concentration range, preferably 35-50%. Volume of Triosyn:100 g. Triosyn size: 500 microns.

FIG. 18 depicts the characteristics of the “F” filter. Characteristicsof the “F” filter: Number of capsule sections: 1. Length of each capsulesections: 6′. Total length of capsule: 6′. Capsule inner diameter:0.75′. Number of diffusers: 0. Triosyn type: iodine component can be30-80% concentration range, preferably 35-50%. Volume of Triosyn: 45 g.Triosyn size: 500 microns.

FIG. 19 depicts the characteristics of the “H” filter. Characteristicsof the “H” filter: Number of capsule sections: 1. Length of each capsulesections: 5′. Total length of capsule: 5′. Capsule inner diameter:0.625′. Number of diffusers: 0. Triosyn type: iodine component can be30-80% concentration range, preferably 35-50%. Volume of Triosyn: 25 g.Triosyn size: 500 microns.

FIG. 20 depicts the characteristics of the “KK” filter. Characteristicsof the “KK” filter: Number of capsule sections: 8. Length of eachcapsule sections: 2.75′ and 1.38′ for the first. Total length ofcapsule: 20.63′. Capsule inner diameter: 0.75′. Number of diffusers: 7.Triosyn type: iodine component can be 30-80% concentration range,preferably 35-50%. Volume of Triosyn: 500 g. Triosyn size: 500 microns.

FIG. 21 depicts the characteristics of the “LL” filter. Characteristicsof the “LL” filter: Number of capsule sections: 5. Length of eachcapsule sections: 2.25′ and 1.31′ for the first. Total length ofcapsule: 10.31′. Capsule inner diameter: 0.625′. Number of diffusers: 4.Triosyn type: iodine component can be 30-80% concentration range,preferably 35-50%. Volume of Triosyn: 45 g. Triosyn size: 500 microns.

FIG. 22 depicts the characteristics of the “0” filter. Characteristicsof the “0” filter: Number of capsule sections: 5. Length of each capsulesections: 2.13′. Total length of capsule: 10.65′. Capsule innerdiameter: 0.625′. Number of diffusers: 4. Triosyn type: iodine componentcan be 30-80% concentration range, preferably 35-50%. Volume of Triosyn:45 g. Triosyn size: 500 microns.

FIG. 23 depicts a top view and cross sectional view of the diffusersused within the filter units/hemoperfusion units. Note that perforateddiffuser affects the flow characteristics of the blood as it passesthrough the unit.

FIG. 24 depicts a battlefield transfusion scenarios using one embodimentof the Triosyn purification system.

FIG. 25 depicts an alternative battlefield transfusion scenarios usingan alternative embodiment of the Triosyn® Purification System.

FIG. 26 depicts a process of cleansing/purifying the blood of virusessuch as the SAR virus and the AIDS virus, within a human using oneembodiment of the Triosyn® Purification System.

FIG. 27 depicts an alterative process of cleansing/purifying the bloodof viruses such as the SAR virus and the AIDS virus, within a humanusing an alternative embodiment of the Triosyn® Purification System.

DETAILED DESCRIPTION OF THE INVENTION

Aspects, features and advantages of the present invention will becomebetter understood with regard to the accompanying description withreference to the drawing figures. What follows are one or moreembodiments of the present invention. It should be apparent to thoseskilled in the art that those embodiments provided herein areillustrative only and not limiting, having been presented by way ofexample only. All the features disclosed in this description may bereplaced by alternative features serving the same purpose, andequivalents or similar purpose, unless expressly stated otherwise.Therefore, numerous other embodiments of the modifications thereof arecontemplated as falling within the scope of the present invention asdefined herein and equivalents thereto. Use of absolute terms, such as“will not,” “will,” “shall,” “shall not,” “must,” and “must not,” arenot meant to limit the present invention as the embodiments disclosedherein are merely exemplary.

1.0 Introduction

The present invention is an innovative hemoperfusion device containing athermal-fused broad-spectrum biocidal iodinated interactive polymer forthe treatment of biological contaminants in body fluids. The developmentof effective decontamination processes active against a broad spectrumof infectious agents could improve the safety level of blood products byeliminating infectious agents that may remain undetected by currentprocedures or new infectious agents for which no screening test has beendeveloped yet. In the context of emergency conditions, of militaryoperations on the battlefield and peacekeeping operations, wheresupplying, conserving and testing of safe blood becomes a realchallenge, the development of a technology that would provide real-timeblood purification when withdrawing blood from a donor would representan important progress.

2.0 Overview of Materials and Methods

The present system employs a Triosyn® thermal fused broad-spectrumiodinated interactive polymer, included in a hemoperfusion column, fordevitalizing high levels of cell-free microorganisms in whole blood andbiological fluids in relation with the characterization of blood cellsviability and function post-treatment.

The biocidal activity of Triosyn® iodinated polymer resin involvingoxidation of microbial cells, post-treatment iodide ions remaining inblood fluid were systematically quantified in order to characterize thecorrelation between the state of cellular integrity and the magnitude ofpotential residual oxidation mechanisms in the treated blood products.

It is known that the white blood cells (WBC) in blood fractions used fortransfusion generally are of no therapeutic benefit to the recipient; anintegrated filter for leukocytes removal was also integrated within thedecontamination system.

The fundamental methodology consisted of: 1) contaminating the blood orbiological fluids with high concentrations (>106 PFU or CFU/ml) of theselected microorganisms; 2) filtering the contaminated products with theTriosyn hemoperfusion unit; 3) sampling and assaying the treatedspecimens for presence of biological contaminants, quantification ofresidual iodide and characterization of the effects of the treatment oncellular integrity.

Tests were performed on bovine whole blood and blood componentscollected in citrate-phosphate-dextrose (CPD) anticoagulant and kept atroom temperature (approximately 25° C.) until the moment of treatment.

2.1 Test Organisms

The organisms used in this procedure are MS2 coliphage (ATCC 15597-B1),Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 25922). Thepropagating host of MS2 is Escherichia coli (ATCC 15597). The MS2coliphage, a bacterial virus known for its survival capabilities in theenvironment, is 23 nm in diameter. Staphylococcus aureus is afacultative anaerobic gram positive enterobacterium with a mean diameterof 0.5 to 1.5 mm. Escherichia coli is a facultative anaerobic grampositive vegetative bacterium presenting 1.1-1.5 mm×2.0-6.0 mm straightrods, occurring singly or in pairs. All the selected organisms showexcellent resistance to environmental conditions and are hardy againstchemical disinfection. They represent good models due to theirnon-pathogenicity, ease of preparation and assay as well as theirstability in stock suspensions.

Test organisms are obtained from an outside source and used to preparesuspensions of the appropriate concentration in order to obtain highchallenge concentrations of 106 PFU or CFU/ml in test matrix to becontaminated. As concerns platelet testing, platelets units werecontaminated with 102 CFU of S. aureus in order to simulate real casescenario concentrations.

2.2 Test System (Purification Process)

The element at the source of the testing system is the Triosynhemoperfusion unit which consists of a low-density polyethylene columnincluding a given volume of Triosyn iodinated biocidal polymer.Throughout the evolution of the research, the first generation ofprototypes was transformed to include a variable number of capsulesections as well as hydrodynamic diffusers. The volume and type ofTriosyn also varied according to the various prototypes. See FIGS. 17-27

A description of the complete integrated blood filtration processdeveloped during this research program is found in Section 3.1.Typically, a volume of blood contaminated with challenge microorganismswas processed through the hemoperfusion prototype to be tested with theassistance of a peristaltic pump at a given flow. Samples were takenafter the filtration of 300-500 ml of blood.

2.3 Controls

Positive controls comprised of CPD anti-coagulated blood, biologicalfluids or platelets were taken from the non-filtered portion of theoriginal volumes. Control samples were subjected to the same storageconditions and testing procedures as the treated samples.

2.4 Flow Rate

For prototype optimization purposes, contaminated biological fluids wereprocessed through the test system at varying flow rates ranging between5 and 250 ml/min. The testing flow rate was controlled with the help ofa Carter™ cassette pump head peristaltic system (Manostat®, BarnantCompany, Illinois, USA) and validated using a stopwatch and a graduatedcylinder. Selected flow rates are specified within the result data.

2.5 Assays

2.5.1 Biocidal Efficacy

The biocidal effect of the treatment was evaluated using standardmicrobiology methods for MS2 phage, S. aureus and E. coli counts. Serialdilutions of the test and positive control samples were plated on MS2specialized agar or Tryptic Soy Agar (TSA) media and incubated forappropriate period and temperature based upon the optimal growthconditions for test organism (12 h at 35.0±0.5° C. for MS2 and E. coli;48 h at 35±0.5° C. for S. aureus) for subsequent enumeration of thenumber of PFU/ml or CFU/ml. The inactivation efficiency was subsequentlyquantified by comparing the number of CFU-PFU/ml recovered from positivecontrols and treated samples. For each test sample, the biocidalreduction was calculated using the following equation:

${B\; R\mspace{14mu} \%} = {\frac{C - T}{C} \times 100}$ Where:C = Control  count.T = Count  for  treated  sample.

2.5.2 Concentration of Residual Iodide

Residual iodide ions in blood fluid were measured for test and controlsamples using an iodide ion selective electrode. Ion selectiveelectrodes (ISE) respond selectively to one species in solution. Theelectrode has a thin membrane separating the sample from the inside ofthe electrode. The internal solution contains the ion of interest at aconstant activity. External solution is the biological fluid. ISEmeasures the potential difference across the membrane, which isdependant on the difference in activity of the iodide species betweenthe internal solution and the biological fluid. Iodide concentration isread directly on the ion meter in millivolts and extrapolated from acalibration curve. Results are expressed in mg/L.

2.5.3 Post-Treatment Effects on Cellular Integrity

Red cell and platelet viability and function were assessed using invitro measurements as predictors of their in vivo recovery and survivalrates. Hematocrit, hemoglobin, blood cells count, mean corpuscularvolume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscularhemoglobin concentration (MCHC) were systematically measured using anautomated blood counter (Vet ABC 45, ABX hematology, Montpellier,France).

Red blood cell potassium was identified by Valeri (71) as a goodindicator of red cell injury during disinfection. Plasma samples werecentrifuged and analyzed for extracellular potassium and sodium dosageusing a flame photometer equipped with a fiber optic cable.

During Phase I of the study, the interpretation of the results wasparticularly based on the methemoglobin (MHb) parameter, because of itssensibility to oxidation. The dosage of methemoglobin usingspectrophotometry techniques measures the conversion of hemoglobin inmethemoglobin which shows an absorption peak at 630 nm. pH was alsostudied as a good indicator of the effect of the treatment on thebiochemistry of the cells and the substrate.

While WBCs are not desired components of blood products for transfusion,they provide easily available data on the quality of treated blood.Consequently, the post-treatment evaluation of WBC viability was used assupplementary information on cell injury associated with disinfectionduring the initial down selection process of first-generationprototypes. The trypan blue exclusion dyeing method with a hemacytometerwas used to evaluate the viability of white blood cells as viewed byphase microscopy.

As far as platelets are concerned, morphological score, pH, total countsand dosage of lactate and lactate dehydrogenase (LDH) enzymes were theparameters considered. As reported by Holme et Al. (1998), the resultsof morphological analysis were found to correlate highly with the extentof shape change (ESC) measured photometrically. In this study, Kunicki'stechnique for evaluating structural changes in platelets was used. The Kscore method is based on morphologic evaluation of platelets as viewedby phase microscopy. Platelet damage is assessed by assigning a score toindividual cells showing distinct morphologic differences. Theconcentration of lactate and lactate dehydrogenase (LDH) enzymes in thesubstrate was measured using colorimetric methods read byspectrophotometry.

2.6 Leukodepletion

A method based on the literature was used for counting leukocytes in redcell products (37, 42, 62 and 77). This counting method describes aprocedure for visual counting of leukocytes present in leukodepletedblood. The method uses a Nageotte counting chamber with 250 ul-grid. Thesensitivity of the method is 0.1 leukocyte/ul and should be used only inproducts for which leukodepletion has reduced the count to levels below5 leukocytes/ul.

3.0 Results and Discussion

3.1 Biocidal Performance

Several factors were found to improve the biocidal performance of bloodconstituent filters having iodinated resins therein by affecting thecontact dynamics within the filters. The fluid dynamics including thevelocity of the blood constituent and the residence time thereof in thefilter as well as all preferential flows and pressure resistancephenomenon were found to have a direct influence on the biocidalperformance of the filters. The elimination of viral particles andbacteria was significantly affected by both the viscosity and the solidcontent of the blood constituent. As evidenced by Table 3.1, thebacteria, which were successfully eliminated in the plasma through theiodinated resin filtration system, seem to be shielded from thedisinfecting agent in the presence of erythrocytes (red blood cells).

TABLE 3.1 Effect of Substrate on E. coli Reduction at ConstantTemperature and Flow Rate E. coli Reduction Filter Type Substrate Avg.Std. Dev. n O blood 56.78 24.56 3 plasma 99.903 0.018 5 BB blood 45.1932.02 6 plasma 99.90 0.14 2

This phenomenon is less evident with MS2 in which case the disinfectionstage possesses a more homogeneous efficacy within the differentsubstrates (blood, plasma or serum) (Table 3.2).

TABLE 3.2 Effect of Substrate on S. aureus Reduction at ConstantTemperature and Flow Rate S. aureus Reduction Filter Type Substrate Avg.Std. Dev. n BB blood 80.62 9.37 6 plasma 99.9986 0.0008 2

Among scientific reviews, certain phenomenon as such are reported. Infact, a few authors (Weber, Barbee, Sobsey and Rutala, 1999) observedthat the presence of blood significantly reduces the antiviralactivities of several chemical agents (sodium hypochlorite, a phenolicand a quaternary ammonium compound). In the light of these facts, webelieve it is reasonable to hypothesize that specific affinities, due inpart to differences in the composition of the membrane as well as theelectric charge of the microorganisms, between certain types of bloodcells or proteinic components and biological agents might be responsiblefor preventing adequate contact with the active ingredient andsubsequent disinfection.

In order to maximize the contact efficacy created within the filtrationsystem, several strategies were tested for different selected prototypesin order to control the parameters influencing the contact betweeniodinated resins and microorganisms. The set-up of hydrodynamicdiffusers was first studied and optimized in order to decrease thesignificance of the variations in the reduction linked to thepreferential flows and the fluctuations of viscosity values. It waspostulated that the addition of diffusers would increase the probabilityof the fluid coming into contact with the iodinated resins, accomplishedin two ways. First, the flow of the blood would be disrupted, causinggreater dispersion of the blood over the available iodinated resinssurface. Secondly, the diffusers would increase the back pressure of theblood, leading to more thorough contact with the iodinated resins beads.The results of subsequent testing suggested that the use of diffuserscontributed to a better control of pressure resistance and a morehomogeneous contact between the polymer and microbial entities with lessvariation observed in biocidal efficacy between similar prototypes.

In order to constantly work toward maximizing the efficacy of the activemicroorganism contact sites, the effects of pre-filtration sonication ofsubstrate and addition of EDTA and calcium chloride (CaCl₂) to thesubstrate in order to prevent the aggregation of microorganisms wereinvestigated. Sonication is contra-indicated for whole blood since itwould induce hemolysis of erythrocytes. The obtained results did notshow a significant effect of these techniques. The sonication of thefiltration system was also examined, the stated hypothesis being thatthe micro-movements created within the filter would potentially increasethe contact opportunities between the existing microorganisms and theactive sites of the polymer. Moreover, the increase in temperature ofthe substrate as an amplification factor of the activity of the polymerwas also studied. The attained results in Table 3.3 allow for observableeffects of sonication and flow rate on the microbiological reductionobtained in the plasma.

TABLE 3.3 A. Effects of Sonication on MS2 Reduction with FilterPrototype JJ Sonification Temperature Flow Rate MS2 Reduction Intensity% Celcius ml/min Avg. % Std. Dev. n 50% 37 25 98.34 1.34 2 50 97.92 — 1100% 37 25 99.54 0.11 2 50 93.15 2.29 2 42 25 99.48 0.05 2

Pursuant to standard operational procedure of transfusion beingselective, it was decided to treat blood components separately (plasma,serum, red blood cell concentrates). In terms of separating thedifferent blood components, a leukodepletion filter was integrated intothe filtration system. Two types of commercial filters were tried(Leukotrap®, Pall Corporation, East Hills, N.Y. 11548, USA andSepacell®, Baxter Healthcare Corp, Il, USA) in different sequences(before or after the Triosyn® unit). In parallel, differentconfigurations were also tested in an attempt to reach the contactequilibrium sought. The optimization of working prototypes entailed thetesting of numerous configurations with regards to composition andstructure of the column (physical dimensions and characteristics of thecolumn, type and amount of polymer, etc.) as well as filtrationconditions (temperature, direction of flow, etc.). The integratedprocedure developed for filtration of red blood cell concentrates readsas follows:

-   -   One unit (500 ml) of fresh whole blood stored at 4° C. for less        than 7 days is brought to room temperature and contaminated with        selected biological agent to a final concentration of 10⁷ CFU or        PFU/ml. The bag is then centrifuged at 3200 RPM for 5 minutes.    -   With the help of a plasma extractor, the plasma is removed from        the bag. 2,75 volumes of phosphate-buffered saline (PBS) are        then added to the bag containing the red blood cell concentrate.        The RBC concentrate is heated up to 37° C. into a thermostated        waterbath.    -   Filtration occurs through the system at a flow of 50 ml/min. The        substrate goes through the Triosyn® unit first to be recuperated        in a transfer bag. The blood is allowed to cool to room        temperature and is centrifuged again at 3200 RPM for 5 minutes.        The PBS supernatant is removed using a plasma extractor.    -   Finally, the red blood cell concentrate is filtrated by gravity        through a Sepacell® filter (Baxter Healthcare Corp, Il, USA) for        leukocytes' removal and recuperated into an adequate bag from        which samples are taken for microbiological assay, hematology        and biochemistry analysis. Control samples are taken from the        whole blood, after contamination; the dilution factor is        accounted for in the reduction calculations.

The development of the Triosyn® integrated system allowed for areproducible biocidal reduction rate greater than 99% against virusesand 99.9% against bacteria in presence of erythrocytes (RBCconcentrates) as demonstrated in the results presented in Table 3.3. Theincrease obtained in terms of microbiological reduction within thissystem is all the more significant since it is associated with aconsiderable reduction of the impacts observed on cellular integrity(methemoglobin and pH levels) as discussed in section below. Theseresults were obtained by maintaining the residual iodide levels wellbelow the limits of the Maximum Tolerated Dose established for largeblood volume transfusions.

TABLE 3.3 Biocidal Reduction for Target Microorganisms in RBCConcentrate Biochemical Parameters pH Methemoglobin Residual IodideMicrobiological Microorganism Filter Type after filtration afterfiltration (%) mg/L Reduction % E. coli N-BB-767 7.03 0.00 221 99.9923N-BB-768 7.17 0.00 331 99.9691 N-BB-769 7.11 0.96 286 99.9844 N-BB-7707.08 1.51 192 99.9897 N-BB-771 7.11 1.18 216 99.9999 S. aureus N-BB-7636.94 1.41 353 99.9122 N-BB-764 6.94 0.00 337 99.9744 N-BB-765 6.89 2.99412 99.9655 MS2 N-BB-759 7.02 0.00 208 99.8062 N-BB-761 7.09 0.00 32599.9744 N-BB-762 7.06 0.65 247 99.9654

Biocidal reduction objectives having been met while maintaining viableblood components, the fourth quarter activities were focused onincreasing the portability and adaptability of the Triosyn® integratedsystem to battlefield conditions. Tests were performed with the intentof replacing the centrifugation steps by alternative blood componentsseparation processes. This would enable the process to take placewithout electrical requirement or peripheral equipment other than theprocessing hemoperfusion system.

The first objective was to remove the centrifugation after the Triosyn®unit from the whole process while maintaining the previously obtainedmicrobiological and biochemical results. Consequently, red blood cellconcentrates were diluted in smaller PBS volumes after the firstcentrifugation step and the Sepacell® filtration was performed at acontrolled constant flow rate.

Results obtained with Escherichia coli showed that the dilution leveland Sepacell® flow rate were not critical for microbiologicalperformance. Conversely, pH and residual iodide were found to beaffected by dilution factors. As shown in table 3.4, tests withcoliphage MS2 demonstrated that the removal of the centrifugation stepaffected the performance of the overall Triosyn integrated system.Moreover, biocidal performance against MS2 fell under the 99% reductionrate. For this reason, other alternatives to replace centrifugationprocess have been considered.

TABLE 3.4 Effect of Dilution of RBCs and Sepacell ® Controlled Flow Rateon Microbiological Efficiency against MS2 Challenge for Selected FilterPrototypes PBS Residual Filter Sepacell ® Dilution MS2 pH iodide TypeFlow Rate Factor n reduction % variation (mg/L) BB 5 1:1 1 99.730 −0.3552 1:2 1 98.756 −0.19 334 10 1:0.5 2 97.922 −0.35 761 1:2 2 85.256−0.18 114 20 1:1 2 95.735 −0.28 349 LL 10 1:0.75 2 98.537 −0.18 485 1:16 96.882 −0.23 504

In order to remove plasma from whole blood, a plasma separator,Plasmaflo® (Asahi Medical Co Ltd, Tokyo, Japan), was used. A plasmaseparator is a medical device used to concentrate red blood cells fromwhole blood by partially removing plasma. Results obtained with thisfilter were compared with those obtained using the centrifugal process.

TABLE 3.5 Comparative Results for Centrifugation versus Plasma Separator(Plasmaflo ®) Processes against Escherichia Coli and MS2 Challenges forFilter Prototypes BB at 50 Ml/Min Residual Methemoglobin pH iodideMicrobiological Microorganism Process n Variation (%) variation (mg/L)Reduction (%) E. coli Centrifugation 5 +0.28 −0.16 249 99.987% Plasmaflo ® 2 +14.34 −0.28 213 >99.9997%   MS2 Centrifugation 3 +0.17−0.16 260 99.56% Plasmaflo ® 2 +9.04 −0.23 260 99.10%

As shown in table 3.5, for similar conditions, methemoglobin levels andpH seemed to be more affected when the substrate was processed throughPlasmaflo® rather than being centrifuged. The fact that more plasmaremained from the Plasmaflo® process probably explains this phenomenonsince it was observed that those parameters show higher values in RBCconcentrates in presence of plasma. Otherwise, residual iodide remainedbelow the Maximum Tolerated Dose limits established previously for largevolume transfusions and biocidal reduction rates were maintained over99% for viruses and 99.9% for bacteria.

Based on these findings, several tests were performed with modifiedfilter prototypes in aim to lessen the negative biochemical effectsobserved with the use of a plasma separator. Those experiments led tothe development of a new Triosyn® system in which two plasma filtersreplaced the previous centrifugation steps proposed in the previousquarter. Table 3.6 shows that up to 99.488% MS2 viral particles wereefficiently eliminated from blood with biochemical parameters close tothose obtained previously, suggesting minimal impacts on cellularintegrity. As concerns bacterial challenge, the improved Triosyn® systemproved to eliminate >99.9997% of E. coli in RBC concentrates (pleaserefer to table 3.5). In addition, in the light of previous observationsregarding the correlation between plasma and methemoglobin levels in RBCconcentrates, it is reasonable to believe that an increase in mechanicalefficiency of the plasma separation process would lead to even lowermethemoglobin levels.

TABLE 3.6 Biocidal Performance of Triosyn ® Integrated System againstMS2 Challenge when Replacing Centrifugation Processes by Two PlasmaFilters Filter Methemoglobin pH Residual iodide MS2 prototype Variation(%) Variation (mg/L) Reduction (%) N-LL-835 +4.83 −0.24 400 99.488N-LL-836 +7.17 −0.18 414 98.815 N-LL-837 +5.24 −0.19 255 98.794 N-LL-838+8.02 −0.22 482 99.388 Average +6.32 −0.21 388 99.121

The modified Triosyn® integrated blood filtration system includingleukocyte removal filters, plasma separators and/or centrifugalprocesses in conjunction with saline or other acceptable physiologicalfluids comprises varying processing sequences in which the order ofelements making up the filtration system may be inverted and flow ratesfluctuated to obtain the most efficient biocidal reduction following theconditions of use. FIG. 1 provides a schematic description of thesystem. The description of pathway A (option A+option A on theschematic), which reads as follows, emphasizes the simplification of theprocedure:

-   -   One unit (500 ml) of whole blood at 37° C. is filtered through a        plasma separator. Plasma is discarded or treated for other use.    -   The red blood cell concentrate (RBC) is diluted to initial        volume using saline or other acceptable physiological fluid at        37° C.    -   The substrate goes through the Triosyn hemoperfusion unit first        and then through a plasma separator at selected flow rate (20        ml/min for instance) to be recuperated in a transfer bag.    -   Finally, RBC is filtrated by gravity through a leukocyte filter        for leukocytes' removal and recuperated into an adequate bag or        immediately transfused.

See FIG. 1. The Triosyn® Integrated Blood Filtration System

The results obtained during the last period show that the modifiedintegrated system offers several alternatives for blood decontamination.Plasmaflo® plasma separator or centrifugation steps might be replaced inthis system by any efficient mechanical plasma separation process, whichallows a great flexibility to the system. In the context of battlefieldoperations, the full process can take place without technologicalsupport, power requirement or specialized training. The collected bloodbag can be connected to the processing filters and the flow could beactivated by a hand pump to achieve full treatment.

3.2 Toxicity Profile

A first step in establishing a toxicity profile for the activeingredient of the filtration system was put in place during the firstquarters of this program. A review and an analysis of the scientificliterature were performed in order to determine the threshold values inview of the elaboration of the Maximum Tolerated Dose (MTD).Toxicological data gathered from the literature suggest an MTD of ioniciodine via intravenous route of 35 mg/kg or 2.5 g for an average humanadult. The following formula was developed to establish the maximumblood volume and filtration time at which the MTD is attained:

Blood Volume_(max)=MTD/Iodide Concentration and FiltrationTime_(max)=Blood Volume_(max)/Flow.

When considering a massive blood transfusion scenario (2.5 litres in anhour for an average human adult), residual iodide concentrations above1000 ppm would be found to exceed the proposed MTD. Conversely, theresidual iodide concentration tolerated varying on to the flow andvolume of fluid transfused, the transfusion of two (2) blood units(2×500 ml) in an average man would allow residual concentrations ofiodide up to 2500 ppm for a filtration time of 20 minutes.

The post-treatment concentration of iodide ions in blood fluids wassystematically quantified in order to characterize the correlationbetween the state of cellular integrity and the magnitude of potentialresidual oxidation mechanisms in the treated blood products. Resultstend to show a correlation between higher values of residual iodide,higher methemoglobin levels and more acid pH in the treated substrate.Based on these results, the assumption was made that there may be anassociation between higher levels of residual iodide and a moreimportant rate of the oxidation phenomenon linked to the amount of sitesthat will be activated on the polymer. The more active sites will beactivated on the polymer, greater the quantity of active species (I₂ ⁺,HOI, I₃ ⁻) that will be released, causing methemoglobin levels to riseas well as a more accentuated acidification of the milieu (substrate).FIG. 2 shows the observed correlation between the residual iodide andmethemoglobin levels.

This hypothesis is consistent with previous findings of our researchsuggesting a connection between the importance of the organic content ofthe substrate to be decontaminated and the amount of sites that will beactivated on the polymer. One can suppose that the presence in thesubstrate of certain elements (namely erythrocytes) having specificaffinities with given microorganisms will hinder effective and rapiddisinfection action by preventing adequate contact with the active sitesof the polymer. Such being the case, the microorganisms still present inthe substrate would keep activating the demand-release mechanism causingmore active complexes (I₂ ⁺, HOI, I₃ ⁻) to leach, resulting in higherconcentrations of residual iodide. The full integrated Triosyn® systemtreating blood components separately allowed the achievement ofimportant biocidal reduction in substrates with high organic contentwhile keeping the residual iodide to low levels. As stated above, theMTD is correlated with the volume of fluid transfused as well as theflow rate of the transfusion. Under the experimental conditionsprevailing for the tests presented in Table 4 (volume=500 ml and flow=50ml/min), residual iodide concentrations of 5000 ppm would correspond tothe MTD proposed in the literature (35 mg/kg). As seen in Table 3.3,greater than 99.9% reduction was obtained against E. coli and S. aureusin RBC concentrates with a mean residual iodide level of 293.5 ppm. Upto 99.97% of MS2 viral particles were eliminated from contaminated RBCwith 325 ppm or below levels of residual iodide. These values representconcentrations well below the limits of the proposed MTD of 35 mg/kgeven when considering large blood volume transfusions at flow ratesapproximating 50 ml/min. FIGS. 3, 4 and 5 demonstrate that, atequivalent biocidal performance, the full Triosyn® system showed lowerconcentration of residual iodide than prototypes selected at the end ofthe preceding period. These results tend to support the hypothesis thattreating blood components separately will enhance contact between themicroorganisms and the active sites of the polymer leading to a swifterdisinfection process and minimal prompting of the demand-releasemechanism.

3.3 Cellular Integrity

For the first year of the project, the main goal aimed at optimizing afirst generation of prototypes able to show a satisfactory biocidalcapacity all by maintaining an acceptable level of integrity for theretransfusion of the treated blood products. Considering the latter, theadopted scientific procedure expounded in previous report was planned in3 steps. The method first consisted of obtaining a satisfactory biocidalreduction while observing the resulting effects of disinfection on thetreated blood products. Secondly, it aimed at selecting and adapting thedesign of the prototypes such as to obtain a maximum reduction for aminimum effect on the cellular integrity. Finally, the third aspect ofthe project would be to consider specific mitigation measures in orderto obtain treated blood products of an optimal quality.

The full integrated Triosyn® system achieves the proposed reductionobjectives while keeping the observed effects on cellular integrity to aminimum.

The in vitro analytical methods are considered as indicators allowingthe prediction, to a certain extent, of the in vivo post-transfusionviability. The retained parameters were selected according to theirrelevance in assessing the disinfection effects of the procedure onblood cells considering the nature of the active ingredient, namely, aniodinated polymer. During Phase I of the study, the interpretation ofthe results was particularly based on the methemoglobin (MHb) parameter,because of its sensibility to oxidation. pH was also studied as a goodindicator of the effect of the treatment on the biochemistry of thecells and the substrate. In standard RBC concentrates, the levels ofmethemoglobin are expected to range between 0 and 2%. Table 3.3 showsthat treatment with Triosyn® integrated system, which eliminated up to99.97% of MS2 viral particles, 99.9999% of E. coli and 99.97% S. aureusbacteria from the substrate, did not affect significantly cellularlevels of methemoglobin allowing normal (between 0 and 2%)post-treatment values to be observed in RBC concentrates. FIGS. 6, 7 and8 underscore the constant decrease in methemoglobin associated with theimprovement of biocidal performance against target organisms asprototype development was progressing. The attenuation of the oxidationprocess of hemoglobin observed when treating RBC alone supports theassumption stated in the previous section that treating blood componentsseparately will enhance contact between the microorganisms and theactive sites of the polymer. A faster elimination of microorganismswould require smaller amounts of the active ingredient for efficientdisinfection to occur.

In other respects, the oxidant nature of the active ingredient likelycontributing to the somewhat acidification of the filtered substrate andprovoking undesirable pH fluctuations, the addition of buffered saline,specifically adapted to the filter environment, to the substrate priorto filtration was proposed to minimize the pH fluctuations. Table 4shows that physiological pH values between 6.89 and 7.17 were observedin RBC concentrates after treatment with Triosyn® integrated systemwhereas Table 3.7 brings out the specific effect of buffered saline inattenuating pH fluctuations.

TABLE 3.7 pH Fluctuations when Adding PBS or 0.85% Saline Buffer Priorto Filtration pH Prototype Before After Buffer Filter filter filter pHvariation Avg. variation Saline 0.85% N-BB-739 7.02 6.68 −0.34 −0.31S-BB-722 7.05 6.62 −0.43 S-BB-723 7.09 6.78 −0.31 S-BB-727 7.15 6.96−0.19 S-BB-735 7.11 6.82 −0.29 PBS N-BB-761 7.26 7.09 −0.17 −0.18N-BB-762 7.22 7.06 −0.16 N-BB-763 7.12 6.94 −0.18 N-BB-766 7.31 7.11−0.20 N-BB-768 7.34 7.17 −0.17

3.4 Platelets

Bacterial contamination of blood components and particularly plateletsis a serious concern for the safety of the blood supply. The majorityopinion in the literature is that platelets stored for the maximum time,5 days, are more likely to be contaminated than those stored for ashorter period.

Bacterial contamination of blood components is the second largest causeof transmission related deaths in the United States, after hemolyticcomplications (50% of deaths), according to the FDA. Bacterialcontamination causes 10% of transfusion related deaths. According toAmerica's Blood Centers, “Bacterial sepsis is the leading microbialcause of transfusion mortality today in the United States—accounting for46 (17%) of 277 reported transfusion deaths from 1990-1998.” (3)

According to the BaCon study, as many as 10% of platelet units arecontaminated with bacteria in the US. It is estimated that 1 in10,000-20,000 units transfused in the US causes a febrile reaction dueto contaminated blood (not specific to platelets) and 1 in 1-6 millionunits results in death. It is believed that significant transfusionreaction occurs once growth reaches the “danger zone”, defined asbacterial concentrations above 10⁶ CFU/ml. (6)

The most common bacteria contaminating platelets are bacteria typicallyfound on the skin, such as Staphylococcus spp., including S. aureus andS. epidemidis. Other bacteria include Salmonella choleraesuis, Serratiamarcescens, and Bacillus cereus. (6) While most contaminations involvegram-positive skin flora, gram-negative bacteria are more frequentlyimplicated in deaths. (3)

While conclusive evidence on how blood products become contaminated hasnot been collected, it is speculated that the major causes are: (6)

-   -   Donor skin at blood donation—either skin is improperly cleaned,        or coring may occur at phlebotomy site.    -   Unapparent donor bacteremia    -   Contamination during collection or processing

In the wake of the tests performed on the different components of blood,a series of preliminary experiments were conducted on the filtration ofplatelet concentrates. Although the current gold standard of clinicalplatelet efficacy is in vivo survival of transfused radio-labelledplatelets, the use of in vitro tests is accepted in the first stages ofR&D as a screening process to eliminate inadequate procedures or developinnovative treatment without requiring human trials (62). Within theseinitial tests, the morphology, pH and bacterial contamination status ofplatelet concentrates were observed before and after procedure. Thetreatment simply consists in processing platelet units (prepared as perstandard procedure and diluted to obtain sufficient volume) through aTriosyn column, as illustrated in FIG. 9, at a flow rate of 10 ml/min.The platelets are directly collected in a transfer bag used for storage.

The following figures allow the observance of the evolution of bacterialload on a timeframe of ten (10) days in the case of filtered vs.non-filtered platelets. These observations were noted on control unitsof platelets as well as units that were contaminated with a strain ofStaphylococcus aureus (10² CFU).

Bacterial growth was detected in 3 platelet units (F1, F3 & F4) in whichthe growth pattern was consistent with cross-contamination due tomanipulation (no sustainable growth over time in the sample, no growthin lower dilutions). However, bacterial load rose to high concentrationsfrom day 3 in one non-treated unit (F3) whereas an upward trend wasdetectable from day 8 in F4 non-treated platelets.

The results show the capacity of the filters in eliminating theinoculated microbial agent such as to prevent further proliferation. Incontrast, an increase in the bacterial load resulting in highconcentrations (10⁸ CFU/ml) of Staphylococcus aureus was noted for allthe non-treated units.

The average curve illustrates the exponential bacteria growth observedin the non-treated platelet units during the 4 days followingcontamination. This growth phase is accompanied by an acidification ofthe medium as evidenced by the pH values presented in table 3.8.Following the exhaustion of the medium, bacteria growth attains aplateau or slightly regresses. On the other hand, no bacterial growthwas observed in the filtered units.

TABLE 3.8 pH Values Observed in Treated and Non-Treated Platelets pH pHpH pH pH Sample Day 2 Day 5 Day 6 Day 7 Day 8 Control F1 7.36 7.53 7.267.18 6.78 F2 7.37 7.57 7.28 7.14 7.12 F3 7.22 7.30 6.90 6.78 6.64 F47.49 7.34 7.15 7.12 7.09 F5 7.40 6.96 6.91 6.90 6.97 F6 7.08 7.07 6.876.91 6.66 Treated F1 7.04 7.75 7.81 7.90 8.05 F2 7.08 7.81 7.91 8.068.26 F3 6.97 7.75 7.80 7.89 7.99 F4 7.16 7.48 7.56 7.77 7.85 F5 7.037.23 7.40 7.53 7.55 F6 7.08 7.34 7.44 7.61 7.68

Table 3.9 presents the morphological score noted following microscopicobservation of the platelets. Overall, the results emphasize thepreservation of the cellular integrity within the filtered plateletswhile the non-treated units present a rapid degradation within theplatelet's morphologic state with the progression of morphologic changesfrom the usual discoid shape to spherical, dendritic and balloon shapes,characteristic of activated platelets.

TABLE 3.9 Changes Noted in Morphological Score of Treated andNon-treated Platelets Mor- phol- Mor- Mor- Mor- Mor- ogical phologicalphological phological phological Sam- Score Score Score Score Score pleDay 2 Day 5 Day 6 Day 7 Day 8 Control F1 B C D D D F2 A D D D D F3 B C CC C F4 B D D D D F5 B D D D D F6 B D D C D Treated F1 A A A A⁻ A F2 A AA A B F3 A B B B B F4 A A A A A F5 A A A A⁻ A F6 A B B B B

Based on FDA recommendations, the lactate dehydrogenase (LDH) andlactate levels in the medium were measured to pursue the in vitroevaluation of platelet biochemistry.

Literature reports that a decrease in platelet count with an increase ofLDH level in the medium can be used as an indicator of platelet lysis.LDH levels and platelet counts were analyzed and compared before andafter treatment over a six-day period. The platelets units have beentreated on day 2 and LDH levels determined before and after treatment onday 2, 3, 4, 5 and 6.

FIGS. 13 and 14 allow the comparison of results obtained from treatedversus control platelet units.

In control samples, the platelet count was found to be relativelyconstant up to day 5. A decrease in platelet count was then observedafter day 6. In parallel, the levels of LDH were found to be constant,and then were followed by an increase on day 5 and a decrease on day 6suggesting the occurrence of platelet lysis on day 5.

The same phenomenon occurred for treated platelet units on day 3 oftesting; specifically, the level of LDH increased on day 3, and thendecreased on day 4. However, despite a significant increase of the LDHlevel on day 5, the platelet count seemed to remain relatively constantuntil the end of storage. Therefore, an increase of the latter cannot becorrelated to platelet lysis. It could, however, be explained by thepresence of an interference. The presence of citrate in the media isoften responsible for interfering with the actual readings of free LDH.

The lactate and pH levels were also determined for treated and controlsamples on day 2, 5 and 6 as seen in FIGS. 15 and 16.

A decrease in the pH level was observed between day 2 and day 5 incontrol samples in parallel with a loss of platelet viability, whichcould explain the critical decrease in platelet counts. Plasma is knownto contain coagulation factors, enzymes and complements. These factorsmay trigger platelet activation and subsequently platelet lysis duringstorage, therefore leading to a decrease in platelet counts. In thatevent, it is not abnormal to observe a decrease of lactate levels overtime. A decrease in platelets is often associated with a glycolysisdecrease, which is then followed by a lactate production decrease.

Usually, an accumulation of lactate due to glycolysis is observed at arate of 2.5 mM/day. A normal increase of lactate levels was observedover time in treated platelets. An initial decrease in the plateletcount was observed the day after treatment occurred to reach a plateauand remain stable throughout the storing process. It was hypothesizedthat this diminution in platelet counts might be reflecting a loss ofactivated platelets, which would be mechanically retained by the filter,contributing to improve the condition of remaining sound platelets overthe storage period.

Using LDH and lactate as testing parameters for in vitro evaluation ofplatelet biochemistry, it is possible to conclude that platelet unitsseemed to be preserved longer when filtrated with Triosyn® units thanthe ones without treatment.

These preliminary findings suggest that the filtration of platelet unitswith an interactive broad-spectrum biocidal polymer may help inpreventing bacterial contamination of platelet concentrates and preservethe integrity of the cells.

3.5 Leukodepletion

Due the possible adverse immune reactions they can cause, white bloodcells (leukocytes) are not desired components of blood products fortransfusion. Thus, embodiments of the Triosyn® filtration systemutilizes leukocyte removal filters, such as those made commerciallyavailable by Leukotrap®, Pall Corporation, East Hills, N.Y. 11548, USAand Sepacell®, Baxter Healthcare Corp, Il, USA. The leukocyte removalfilter may be situated within the Triosyn® filtration system indifferent sequences (before or after the Triosyn® unit).

The manufacturers of Leukotrap® and Sepacell® filters guarantee a 99.95%reduction of leukocytes in the blood. The reduction standard based onapplicable federal statutes and regulations of the Food and DrugAdministration (FDA), US Department of Health and Human Servicesrequires leukodepleted products to show <5×10⁶ leukocytes/unit counts.The effectiveness of leukocyte removal under the actual conditions ofuse and the physical configuration of the decontamination system had tobe demonstrated.

The counting of leukocytes in red cell products (34, 36, 54 and 63) isbased on a conventional method. This counting method describes aprocedure for visual counting of leukocytes present in leukodepletedblood. The method uses a Nageotte counting chamber with 2 50 ul-grid.The sensitivity of the method is 0.1 leukocyte/ul and should be usedonly in products for which leukodepletion has reduced the count tolevels below 5 leukocytes/ul. See Table 3.10.

TABLE 3.10 White Cell Counts in Red Blood Cells Leukodepleted withSepacell ® Filters No Nutricell added WBC 100 ml Nutricell added beforeWBC after WBC after Sepacell Sepacell % Sepacell % Sample cells/Lcells/L reduction cells/L reduction N-BB-769 1.06E+10 2.41E+07 99.777.48E+06 99.93 N-BB-770 8.10E+09 1.49E+07 99.82 3.53E+06 99.96 N-BB-7717.10E+09 2.67E+07 99.62 7.50E+06 99.89 021216-37-4 6.30E+09 8.10E+0699.7 3.20E+06 99.95 021216-61-7 7.30E+09 8.00E+06 99.89 2.80E+06 99.96

Advantages

Some advantages and accomplishments of the present invention include thefollowing:

-   -   Demonstrated reproducible biocidal capacity exceeding 99.9% in        the eradication of MS2 viral particles, E. coli and S. aureus        bacteria from RBCs suspensions.    -   Up to 99. 97% reduction of MS2 viral particles in red blood cell        concentrates with maintenance of normal methemoglobin level and        physiological pH.    -   Up to 99.9999% reduction of E. coli concentration in red blood        cell concentrates with maintenance of normal methemoglobin level        and physiological pH.    -   Up to 99.97% reduction of S. aureus concentration in red blood        cell concentrates with maintenance of normal methemoglobin level        and physiological pH.    -   Achievement of >99.9% biocidal reduction (including viruses and        bacteria) while keeping residual iodide levels well below the        limits of the Maximum Tolerated Dose (35 mg/kg) considering a        massive transfusion scenario (2.5 L in an hour).    -   Prevention of bacterial proliferation in platelet concentrates        with preservation of cellular integrity.    -   Increased storage time (up to 10 days) of platelets without        bacterial degradation.    -   Experimental demonstration of elimination of Staphylococcus        aureus inoculated in platelet samples with no observable adverse        affects.    -   Integration of effective leukocyte depletion to the filtration        system according to FDA standard (<5×10⁶ leukocytes/unit).    -   Improvement of portability and adaptability of Triosyn® Triosyn        integrated blood filtration system while maintaining biocidal        performance and minimal impact on cellular integrity.

CONCLUSION

Considering the variations observed in the biocidal reduction accordingto the type of substrate filtrated and based on the fact that bloodcomponents are usually administered separately in current transfusionpractices, an integrated Triosyn® filtration system was developed forseparate treatment of blood components. The integrated system includescommercially available leukocyte filters which proved to be efficientunder the actual conditions of use and the physical configuration of thedecontamination system. During previous quarters, reduction objectivesof 99% were exceeded in plasma and serum with, respectively, up to99.9978% and greater than 99.9999% reduction of MS2 viral particles andE. coli. The development of the integrated system allowed for areproducible biocidal reduction rate greater than 99.9% against MS2viral particles and S. aureus bacteria in presence of erythrocytes (RBCconcentrates) whereas bacterial reduction of up to 99.9999% was obtainedagainst E. coli. The increase obtained in terms of microbiologicalreduction within this system is all the more significant since it isassociated with a considerable reduction of the impacts observed oncellular integrity. Normal levels of methemoglobin (between 0 and 2%)and physiological values of pH were maintained in RBC concentrates aftertreatment with Triosyn® integrated system. The examination of theprogression of biocidal reduction versus residual iodide levels in bloodover the last 6-month testing period showed that, for equivalentbiocidal performance, the full Triosyn® system showed lowerconcentration of residual iodide than prototypes selected at the end ofthe preceding period, i.e. values representing concentrations well belowthe limits of the proposed MTD (325 ppm or below vs 5000 ppm threshold).

Detail of Triosyn Filter Unit

FIGS. 4-9 depict exemplary embodiments of different types of filterunits/hemoperfusion units (namely BB, F, H, KK, LL and O, respectively)that may be used within the present invention. The specificcharacteristics of both the column and the iodinated polymer for eachtype of filter are included in the respective drawings.

Exemplary Process Flows

In accordance with the present invention, the following section entitled“Triosyn Blood Technology” describe integrated processes forpurification of blood and blood components using iodinated resin,leukocyte removal filters, fluid separators and/or centrifugal processesin conjunction with saline or other acceptable physiological fluids.Processing sequences may be varied, that is, the order of elementsmaking up the filtration system may be inverted and flow ratesfluctuated to obtain the most efficient biocidal reduction following theconditions of use.

Application

FIG. 23 depicts a top view and cross sectional view of the diffusersused within the filter units/hemoperfusion units. FIGS. 24 and 25 depictalternative battlefield transfusion scenarios using two embodiments ofthe Triosyn purification system. FIGS. 26 and 27 depict processes ofcleansing/purifying the blood of viruses such as the SAR virus and theAIDS virus, within a human using two embodiments of the Triosynpurification system.

Appendices

The accompanying Appendices also form part of this disclosure.

1. A method for decontaminating blood, comprising: a) providing ahemoperfusion unit containing a low-density chamber including aneffective amount of demand disinfectant, said demand disinfectant beingan iodinated resin; b) separating a unit of blood infected with aquantity of bacteria or viral particles to provide a red blood cellconcentrate and a plasma concentrate; c) discarding the plasmaconcentrate; d) diluting the red blood cell concentrate with an aqueoussolution; e) filtering said diluted red blood cell concentrate throughsaid hemoperfusion unit at a selected flow rate, wherein the amount ofbacteria or viral particles is reduced by greater than 90%.)
 2. Themethod of claim 1, wherein the amount of bacteria or viral particles isreduced by greater than 95%.)
 3. The method of claim 1, wherein theamount of bacteria or viral particles is reduced by greater than 99%.)4. The method of claim 1, wherein the bacteria or viral particles areselected from the group consisting of Staphylococcus aureus, E. coli andMS2.)
 5. The method of claim 1 further comprising gravity filtering redblood cell concentrate through a leukocyte filter to remove leukocytesprior to diluting the red blood cell concentrate.)
 6. The method ofclaim 1 further comprising gravity filtering red blood cell concentratethrough a leukocyte filter to remove leukocytes after diluting the redblood cell concentrate.)
 7. The method of claim 1, wherein the aqueoussolution is a saline solution.)
 8. The method of claim 1, wherein thesaid flow rate is 50 ml/mm.)
 9. The method of claim 1, wherein the saidflow rate is 20 ml/mm.)
 10. The method of claim 1, wherein the alow-density chamber further contains hydrodynamic diffusers.